Cottonseed is a valuable product of cotton. Cottonseed contains approximately 25% of cottonseed oil, a well established commodity vegetable oil because of its use as either a food ingredient or as a cooking oil for food preparation (Cherry, 1984). The world production of cottonseed oil in 1997/98 was around 4 million tonnes, making it sixth in importance behind the oils of soybean, oil-palm, rapeseed, sunflower and groundnut (Oil World Annual. 1998).
Globally, cotton crops consist of four domesticated Gossypium spp., including the allotetraploid species G. barbadense L. and G. hirsutum L., and the diploid species G. arboreum L. and G. herbaceum L. Of these four species, G. hirsutum (upland cotton) is the predominant species, accounting for the overwhelming majority of cotton production worldwide. Currently, most cottonseed oil is derived from G. hirsutum, possibly as a consequence of the fact that this species is the major worldwide source of cotton fibre, and, cotton crops are primarily grown for their fibre.
The major components of cottonseed oil are the saturated fatty acids, palmitic acid (C16:0) and stearic acid (C18:0); the monounsaturated fatty acid, oleic acid (C18:1); and the polyunsaturated fatty acids, linoleic acid (C18;2) and α-linolenic acid (C18:3). A typical cottonseed fatty acid profile contains high levels of palmitic acid (24%) and linoleic acid (54%), a moderate level of oleic acid (18%). and a very low level of stearic acid (3%) and α-linolenic acid (1%).
The number, position, and conformation of a double bond in each fatty acid present in the cottonseed influences the physical properties (such as melting temperature), chemical properties and nutritional value of the cottonseed oil, and the applications to which it may be put, particularly in the food industry.
For example, the presence of a carbon double bond in a monounsaturated fatty acid or polyunsaturated fatty acid lowers its melting temperature, compared to the melting temperature of a saturated fatty acid of the same carbon chain length, such that the C-18 unsaturated fatty acids, oleic acid, linoleic acid, and linolenic acid, are all liquid at ambient temperature.
Additionally, the susceptibility of a fatty acid to oxidation increases proportionately with the number of carbon double bonds present in the fatty acid molecule, dramatically reducing the suitability of oils containing polyunsaturated fatty acids to applications involving the use of prolonged heat in the presence of oxygen, such as cooking and other food service applications.
For applications that require solid fat components such as in solid cooking fats, shortenings, or margarines, it is necessary to have moderately high levels of saturated fatty acids, or the functionally equivalent trans-fatty acids. Trans-fatty acids have carbon double bonds in the trans-orientation rather than the naturally-occurring cis-orientation.
Currently, the unsaturated fatty acids are subjected to chemical hydrogenation, to improve their suitability in cooking and food service applications. Hydrogenated cottonseed oil is a valuable product, because cottonseed oil has a naturally-high level of palmitic acid, and desired melting properties can be readily achieved by the hydrogenation process. In this process, trans-fatty acids are produced as an artifact.
The nutritional quality of natural cottonseed oil, and hydrogenated cottonseed oil, has been questioned because of the reported adverse effects of both saturated fatty acids, and trans fatty acids (Wollett 1994). The contribution of high levels of some saturated fatty acids in the diet, particularly palmitic acid, to increased blood cholesterol, and more particularly to increased low density lipoprotein (LDL), is well-established. Elevated LDL in the blood has been associated with an enhanced risk of cardiovascular disease in humans. Moreover, trans-fatty acids also elevate LDL cholesterol in a manner similar to palmitic acid. Because of the proven association with risk of cardiovascular disease, nutritionists and health authorities generally recommend limiting the dietary intake of palmitic acid, and trans-fatty acids, to at least below 30% of total fat intake. Natural oils high in palmitic acid, and hydrogenated oils high in trans-fatty acids, are expected to lose favour as a consequence of these recommendations.
However, not all saturated fatty acids are associated with elevated cholesterol. For example, stearic acid is reported to have neutral effects on blood cholesterol (Wollett 1994). in this respect, the high melting temperature of stearic acid (approximately 70° C.) also makes it particularly suitable in solid fat applications. Accordingly, because of its neutral effects on blood cholesterol levels, a high stearic acid-containing oil is a desirable substitute for partially-hydrogenated plant oils currently used in margarine production. Because of its physicochemical properties, stearic acid is also suitable for use in the production of cosmetics, pharmaceuticals and candles (Topfer, 1995). Furthermore, novel cottonseed oil having approximately equal proportions of palmitic, stearic and oleic and therefore having considerable potential for use as a cocoa butter substitute.
Although polyunsaturated fatty acids are beneficial in terms of lipoprotein metabolism and cardiovascular health, they are highly susceptible to peroxidation.
In summary, because the major use of cotton seed oil is as a foodstuff, there is a need to develop improved oils that have enhanced human nutritional value, such as, for example, oils that have reduced palmitic acid and/or are high in stearic acid. Furthermore, there is also a need to improve the potential applications of cottonseed oil in the food industry, without the adverse health risks associated with using oils rich in many saturated fatty acids, by providing oils having novel unsaturated fatty acid profiles. For example, oils having high oleic acid content and/or low linoleic acid content have the desired physicochemical properties of a cooking oil, and do not require hydrogenation (Kinney 1996).
Furthermore, triacylglycerol composed of approximately equal proportions of palmitic acid, oleic acid, and stearic acid (i.e. POS-type triacylglycerol) has a very sharp melting temperature at around body temperature, making it particularly suitable as a substitute for cocoa butter in the manufacture of chocolate and other confectionery. (Fincke, 1976; Gunstone et al., 1986). Currently, the seeds of the cocoa tree, Theobroma cacao L., are the sole source of cocoa butter, and, as a consequence, cocoa butter is often in short supply and costly. The development of cocoa butter substitutes is of considerable economic importance.
In some cases, plant breeders have been able to modify the fatty acid content or composition of seed-derived oils, by inducing mutations in fatty acid biosynthesis genes. Exposure of plant material, generally seeds, to certain mutagenic agents, such as radiation or chemical mutagens, combined with traditional plant breeding approaches, has successfully produced a wide range of novel fatty acid profiles in many oilseed crops, including mutants of rapeseed (Auld et al., 1992), sunflower (Soldatov, 1976) and soybean (Rahman et al., 1994), having increased oleic acid; mutants of soybean (Erickson et al., 1988), linseed (Rowland and Bhatty, 1990), and sunflower (Osorio et al., 1995), having increased palmitic acid; mutants of soybean (Fehr et al, 1991) having lowered palmitic acid; mutants of soybean (Graef et al. 1985: Rahman et al., 1995) and sunflower (Osorio et al., 1995)having increased stearic acid; and mutants of linseed (Green. 1986), soybean (Wilcox et al., 1984), and rapeseed (Robbelen and Nitsch, 1975) having lowered linolenic acid. In several cases this has led to the commercial exploitation of these mutants, such as in the development of commercial varieties of high-oleate sunflower (Miller et al., 1987) and low-linolenic linseed (Green et al. 1991) and rapeseed (Scarth et al., 1988) oils. However, in spite of such notable successes in most other oilseed crops, there are no reports of substantial genetic modification of fatty acid composition in cottonseed oil using induced mutagenesis.
As a result of extensive basic biochemical research over a number of decades, the pathway for synthesis of the predominant fatty acids and their subsequent assembly into the seed storage triglycerides (oils) is now well understood for many plant species. Nearly all of the enzymes involved in fatty-acid metabolism have been identified, the biosynthetic steps catalysed therefor characterised, and the genes encoding said enzymes cloned. In particular, the genes encoding stearoyl-ACP Δ9desaturases, and oleoyl-ACP Δ12 desaturases have been cloned from several oilseed species, as follows.
The cDNAs encoding fatty acid Δ9-desaturase (Δ9 stearoyl-ACP desaturase) enzymes from approximately 22 plant species, including castor bean (Shanklin and Somerville 1991), safflower (Thompson et al., 1991), and cotton (Liu et al., 1996) have been cloned, and the nucleotide sequences thereof made publicly available from the GenBank database. Antisense gene constructs comprising a nucleotide sequence complementary to the Brassica rapa stearoyl-ACP Δ9-desaturase cDNA have been used to decrease expression of the endogenous B. napus and B. rapa stearoyl-ACP Δ9-desaturase genes (Knutzon 1992), thereby increasing stearic acid at the expense of oleic acid in the seed oil. In this case, stearic acid was increased to 40% of total fatty acid in the seed.
With regard to fatty acid Δ12-desaturase genes, a cDNA containing the open reading frame of the Arabidopsis thaliana FAD2 gene has been isolated, and shown to complement the fad2 mutation of A. thaliana, which mutation produces a deficiency in the activity of the oleoyl-PC Δ12-desaturase enzyme (Miquel and Browse, 1992), indicating that the FAD2 gene encodes an oleoyl-PC Δ12-desaturase (Okuley et al., 1994). Kinney (1997) decreased expression of endogenous rapeseed and soybean fatty acid Δ12-desaturase genes, by using sense-suppression (cosuppression) and antisense-suppression gene constructs, to produce high oleic acid-containing oils. In that work, Kinney (1997) also reported the decreased expression of fatty acid Δ15-desaturase genes, to produce low linolenic acid-containing oils in both rapeseed and soybean. Cosuppression to reduce expression of an endogenous fatty acid Δ12-desaturase gene has also been reported to produce high oleic acid oils in Brassica napus and Brassica juncea (Stoutjesdijk et al., 1999).
U.S. Pat. No. 5,850,026 (Cargill, Inc.), dated 15 Dec. 1998, also reports the production of high oleic acid-containing oilseed in Brassica sp., by using antisense or cosuppression gene constructs directed simultaneously against microsomal fatty acid Δ12-desaturase and microsomal fatty acid Δ-15 desaturase gene expression. The oilseed reported by these workers was also low in erucic acid and α-linolenic acid.
U.S. Pat. No. 5,981,781 (E.I. du Pont de Nemours and Company), dated 9 Nov. 1999, teaches the use of cosuppression, to reduce expression of the soybean GmFAD2-1 gene, which encodes a fatty acid Δ12-desaturase (oleoyl-PC Δ12-desaturase) in that species. A high oleic acid-containing soybean oil, having high oxidative stability, was produced by this cosuppression.
More recently, Liu et al. (1999a, 1999b) have described a fatty acid Δ12-desaturase (oleoyl-PC Δ12-desaturase) gene from cotton.
Notwithstanding the considerable number of publicly-available plant fatty acid biosynthesis genes which have been cloned and characterised,; and the reported modification of fatty acid levels in the oils of Brassica spp. and soybean using said genes, there is no reported modification of fatty acid metabolism in cotton, using either traditional plant breeding, mutational breeding, or recombinant DNA approaches. The tetraploid nature of cotton, and the existence of large families of specific fatty acid biosynthesis genes makes it difficult to determine those genes which, by virtue of being expressed in a seed-specific manner are suitable targets for silencing with a view to modifying oil seed composition.
Additionally, gene silencing is not a straightforward procedure as applied to cotton. There are only a few reports in the literature of the transformation of cotton using gene silencing gene constructs, and these reports are restricted to the use of antisense technology. For example, antisense technology has been used to reduce the expression of genes involved in fibre synthesis, however in that report the transgenic plants did not exhibit a detectable phenotype notwithstanding a reduction in enzyme biosynthesis, suggesting that the silencing of genes in cotton is unpredictable